Ovarian tissue cryopreservation (OTC) is very debated in women with breast cancer, chemotherapy and mutations in breast cancer 1 and breast cancer 2 (BRCA) genes, due to the high risk of developing ovarian cancer after ovarian tissue reimplantation. Objective: The present study aims to evaluate the effects of a double slow freezing/rapid thawing procedure on the quality of human ovarian tissue, in terms of preservation of tissue components and follicle viability by light microscopy. Methods: To this end the ovarian tissue of three women, donated for research purposes, was subjected to a single (T1) and double (T2) OTC procedure. For each woman fresh (CTRL-T0), T1 and T2 samples were fixed in formaldehyde for light microscopy analysis. In addition, for T1 and T2 samples of each woman an additional sample was thawed, in vitro cultured for 4 hours (h) and subsequently subjected to neutral red staining combined with histological evaluation. Results: After re-freezing, a good preservation of follicles and stroma was observed by histological and immunohistochemical (Ki67 and Bcl2) analysis. Moreover, a high follicle survival rate resulted after in vitro culture and Neutral Red staining followed by histological evaluation. Discussion: Although the analyzed tissue was not reimplanted in human or/and animal models, these preliminary data, suggest the possibility of twice freezing ovarian tissue as a potential new way to treat women with breast cancer at risk of recurrence or ovarian cancer. In these women to safeguard the women’s health, the transplanted ovarian tissue could be removed after childbearing and re-frozen for later motherhood request.